Project Summary Inflammatory breast cancer (IBC) is one of the most aggressive and lethal form of breast cancer. The molecular mechanisms underlying IBC are poorly understood. Therapeutic strategies have been adopted from non-IBC breast cancers and even though outcomes have been improved for HER2-positive and hormonal receptor IBC, prognosis for triple-negative (TN) subtype IBCs (ER-/PR-/HER2-) is still dire. Currently, estrogen non-genomic signaling has been associated with progression, motility, and invasion of TN breast cancer and most recently of IBC. This proposal seeks to elucidate the functional role of estrogen signaling in IBCs and to compare the effects of estrogen in two contexts: HER2-positive and triple-negative IBC subtypes. Analyses will be performed to define the effects of estrogen signaling, regulated by ER?36 and GPR30, and the cross- activation of ErbB signaling important in the acquisition of various oncogenic phenotypes. The target cells for analysis will be IBC cell lines, SUM149 (TNBC) and SUM190 (HER2+). Around 40% of IBC are TN breast cancers making it hard to treat with endocrine therapy, which is quite effective in ER+/PR+ BC subtypes. IBC shows over-expression of ErbB tyrosine kinase receptors, especially EGFR, in about 50% of the cases, but targeted therapy against this receptor has not been effective for this disease. A series of mechanistic studies will be performed to evaluate the effects of estrogen in the acquisition of oncogenic phenotypes upon knockdown or over-expression of the alternative estrogen receptors, and activation or inhibition of the estrogen signaling. IBC cell lines will be treated with ligands and inhibitors (estradiol,G1,G15, Icaritin) specific to the estrogen receptors to then thoroughly characterized the activation of EGFR downstream kinases and expression of effector proteins and transcriptional changes associated with estrogen signaling. After identifying kinases activated by estrogen signaling, drug response analyses will be done to test novel therapeutic targets with single agents or in combination with EGFR inhibitors. The effects of estrogen non-genomic signaling in pro-oncogenic phenotypes (proliferation, motility, and invasion) will be evaluated using a novel invasion assay and three-dimensional culture system that mimics the formation of IBC tumor emboli. Also, we will determine the DNA binding preferences of ER?36, due to the fact that this isoform of ER? conserved the ligand binding domain, DNA binding domain and nuclear localization signal. In parallel, by RNA-seq analysis we will determine the transcriptome changes associated with estrogen treatment in our two cell line models to identify pathways affected by estrogen relevant in motility, invasion and/or stemness. The poor prognosis for patients with IBC emphasizes the need to further characterize the functional changes associated with its aggressive progression and the interaction of estrogen non-genomic signaling with EGFR pathway. In the long-term, this study will help us design more effective targeted therapies that can be tested in animal models and understand further the mechanisms associated with IBC progression.